Biosynthesis, characterization and anthelmintic activity of silver nanoparticles of Clerodendrum infortunatum isolate

Over the past few decades, the green synthesis of nanoparticles has gained importance for their therapeutic efficacy and eco-friendly nature. Integrating green chemistry principles into multidisciplinary nanoscience research has paved the way for developing environmentally benign and sustainable methods for synthesizing gold and silver nanoparticles. In the present study, the flowers obtained from Clerodendrum infortunatum (L.), belonging to the family Verbenaceae, have been used for biosynthesizing silver nanoparticles (AgNPs) to evaluate the anthelmintic potential. UV–Vis spectroscopy, XRD, FTIR, SEM and TEM analyses were performed to ascertain the formation of AgNPs. Clerodendrum-derived AgNP (CLE-AgNP) has significantly affected the normal physiological functions of the poultry parasite Raillietina spp., a menace to the livestock industry. Our study manifests that CLE-AgNPs cause considerable distortion of the surface tegument of this cestode parasite leading to changes in the host-parasite interface. The histochemical localization studies of the tegument-associated enzymes viz. AcPase, AlkPase, ATPase and 5’-Nu, exposed to the drug, showed a substantial activity decline, thus establishing the anthelmintic potential of the CLE-AgNPs.


Results
Qualitative assessment of phytochemicals. The therapeutic properties of the CLE flower can be associated with the presence of secondary metabolites such as alkaloids, flavonoids, saponins, phenols and tannins. Table 1 shows the phytochemical analysis of the aqueous extract from the flowers. These phytochemicals are vital in the reduction, capping, and stability of generated AgNPs, which also aid in averting nanoparticle agglomeration and enhancing their biological activity.

UV-visible (UV-Vis) spectrophotometric analysis. AgNPs exhibit a strong absorption band and
generate specific color in solution due to the surface plasmon resonance (SPR) 39 . The colorless AgNO 3 solution became pale yellow after two hours and turned dark brown after 12 h of incubation at room temperature (Fig. 1a). The dark brown color observed at the end of the synthesis might occur at the 400-500 nm wavelength range due to the stimulation of surface plasmon vibrations specific to AgNPs 40 . The reduction of Ag + ion to Ag° during the reaction with the ingredients in CLE extract was observed by UV-Vis spectroscopy for the 350-1100 nm wavelength range. The magnitude of the absorption peak increased with reaction time due to the increased number of AgNPs. The maximum absorption of the CLE-AgNP sample was found at 405 nm (Fig. 1a), which confirmed the formation of the desired AgNPs.
Fourier transform infrared spectroscopy (FTIR) analysis. FTIR spectra were recorded to identify probable functional groups of the biomolecules present in CLE flower aqueous extract responsible for forming and stabilizing CLE-AgNPs ( Fig. 1b and c). The strong bands in Fig. 1b at 3418, 2922, 2361, 1647, 1541, 1383, 1261, 1028, 808, and 669 cm −1 corroborate with the capping agents responsible for the AgNPs formation. The vibration band at 3418 cm −1 in the spectra is assigned to O-H stretching in alcohols and phenols. The peaks near 2912 and 2361 cm −1 correspond to C-H and C-O or N-H stretching vibrations, respectively. The strong peak at  (Fig. 2a). These peaks correspond to the crystal planes (111), (200), (220), and (311), respectively and match with the powdered diffraction standard values of Miller indices (hkl) of silver 40 . The diffraction angles corresponded to the face-centered cubic (FCC) structure of silver in AgNPs and agreed with the standard powder diffraction card of the Joint Committee on Powder Diffraction Standards (JCPDS) corresponding to silver (File No.: 04-0783) 42 . The average crystal size D of the AgNPs has been estimated from the diffractogram by using the Debye-Scherrer formula, D = 0.9 /βCosθ , where is the wavelength of the X-rays used for diffraction, θ the Bragg angle, and β the full width at half maximum (FWHM) of a diffraction band 43 . Based on the XRD spectrum of the CLE-AgNP, the average crystal size of the nanoparticles was calculated to be 27.67 nm.
Scanning electron microscopy (SEM) studies. The morphology of CLE-AgNPs characterized using SEM, revealed granular and spherical-shaped particles with diameters less than 50 nm, as shown in Fig. 2b. Some particles in the SEM image appear bigger than the average size due to nanoparticle aggregation caused by solvent evaporation due to critical point drying during the sample preparation.
Transmission electron microscopy (TEM) studies. The dispersion, aggregation, crystalline state and size of the CLE-AgNPs were examined using TEM, as shown in Fig. 2c. The sizes of CLE-AgNP particles were measured using ImageJ (https:// imagej. nih. gov/ ij/), and a histogram of sizes was calculated. The Gaussian fitting of the histogram yielded the mean particle size of CLE-AgNPs to be 27.75 ± 3.13 nm (mean ± sd, N = 90) (Fig. 2d). The surface morphology studied by TEM showed clear evidence for the metallic crystal formation of CLE-AgNPs, which was dispersed uniformly with fewer particle aggregation, and the particles formed in a spherical shape. The presence of some larger nanoparticles may be attributed to the fact that CLE-AgNPs tend to aggregate due to their high surface energy and high surface tension of the ultrafine nanoparticles 44 .
Efficacy of CLE-AgNP on Raillietina spp. Raillietina spp., incubated in the control medium (PBS only), showed physical activity for a longer period; the controls survived for about 72.00 ± 0.04 h, following which they became immobilized and dead (Fig. 3). On exposure to the test medium CLE-AgNP and Genistein (reference drug), the parasites proceeded from the vigorous movement state to the relaxed state, following which they attained paralysis leading to death. The in vitro evaluation of the efficacy of CLE-AgNPs against the cestode  , and 125 µg/ml, respectively. We observed reduced anthelmintic effects on the cestode parasite when exposed to CLE-aqueous extract only (Supplementary Table S1).
Morphological changes of CLE-AgNP-exposed Raillietina spp. SEM images of the control parasites reveal a rostellum and four suckers arranged sideways around the scolex, each with circlets of broad hooks at the bottom, tapered, and bent toward the ends (Fig. 4a). At higher magnification, the surface tegument of the proglottid is revealed to be covered with smooth, homogenous microtriches, which are the absorptive structures  www.nature.com/scientificreports/ for feeding (Fig. 4d). However, on exposure to the test medium, the general surface topography of the proglottids degenerated with the formation of wrinkles (Fig. 4f). The cestode treated with CLE-AgNP showed irrevocable destruction of the scolex, which appeared greatly distorted with suckers extensively shrunken and sharply crooked spines around the suckers. The filamentous nature of microtriches was lost, and the spines around suckers were broken and eroded (Fig. 4c), which altered the maintenance of the parasite position on the host cell, affecting its nutrition. The Genistein-treated parasites showed immense disfigurement of the scolex, breakage and disembarkment of the tegumental surface structures (Figs. 4b, e).
Histochemical studies. The tegument (T) of the control Raillietina spp. showed intense activity of Acid phosphatase (AcPase), Acid phosphatase (AlkPase), Adenosine triphosphatase (ATPase) and 5'-Nucleotidase (5'-Nu) compared to sub-tegument (ST) and somatic musculature (SM) (Fig. 5a-d). Figure 5e-h depict the histological sections of CLE-AgNP exposed parasites showing a general reduction in the staining intensity in the T, ST and SM, whereas the parenchyma cells (P) remained unaffected. AcPase showed a pronounced reduction in stain intensity in the T and ST region for the AgNP incubated section (Fig. 5a,e), indicating disruption of the parasitic membrane transport metabolism. However, there was minimal activity throughout the section of parasites exposed to Genistein (Fig. 5i-l). A much-diminished activity of AlkPase was observed throughout the treated sections of the parasite (Fig. 5b,f). The ATPase activity was almost imperceptible in the T and ST of the parasite treated with CLE-AgNP compared to the control parasites (Fig. 5c,g). The 5'-Nu activity was also found to be reduced throughout the T and ST region in the AgNP exposed cestodes compared to the control (Fig. 5d,h). The decrease in the enzymatic activities resulted from the tegumental damages caused by the CLE-AgNPs.

Discussion
We utilized Clerodendrum flowers for the extracellular production of silver nanoparticles and demonstrated their capabilities as an alternative to synthetic chemical techniques. In recent times, the synthesis of nanoparticles from natural sources has gained immense publicity. Organic nanoparticles such as chitosan and lipid nanoparticles and inorganic nanoparticles such as gold have been used as nano-drug delivery systems [45][46][47][48][49] . The mode of bactericidal effect of nanoparticles has not yet been fully elucidated. However, it irreversibly disrupts the bacterial cell wall, inactivates vital proteins, chelates DNA, and forms reactive oxygen species known to have high microbicidal activity 41,[49][50][51][52] . While microorganisms continue to be investigated for metal nanoparticle synthesis, using plant extracts in the biosynthesis of nanoparticle manufacturing processes is an attractive prospect that is yet largely unknown and underutilized. In the current investigation, we focused on the green synthesis of CLE-AgNP by adding plant extract to the AgNO 3 solution (Fig. 6a), which was initially confirmed by the color changes of the solution from colorless to pale yellow and later measured spectrophotometrically (Fig. 1a). The intensity of www.nature.com/scientificreports/ the color increased in direct proportion to the incubation period, and the highest absorption peak indicated the maximal yield. Previous research has indicated that plants contain phenols and flavonoids with great antioxidant functions, enabling the synthesis of nanoparticles 53,54 . Evidence identifying various phytoconstituents in C. infortunatum flower screened for flavonoids, saponins, phenol, and tannins with potential therapeutic pursuits rationalized our objective for preparing CLE-AgNPs. Although the exact underlying mechanism for forming AgNPs is unclear, it has been proposed that the various active functional groups of phytochemicals in CLE flower are responsible for biosynthesis AgNPs by reducing Ag ions to their elemental form 55,56 (Fig. 6b). Bioreduction of silver ions is followed by nucleation and growth of the adjacent reduced silver atoms into silver nanoparticles of characteristic shape and size. Finally, the newly synthesized nanoparticles are stabilized by capping with the phytochemicals. The capping agents were confirmed by the FTIR analyses of the nanoparticles. For CLE-AgNPs, we observed that most of the functional groups present in the flower aqueous extract were intact in the synthesized nanoparticle. The absorption bands mainly represented the presence of functional groups such as phenol, ketones, carboxylic acids, amines and amides. Compared to the CLE flower extract, the FTIR measurements of CLE-AgNP showed the disappearance of peak at 1541 cm −1 , indicating that the polyols like flavonoids and terpenoids in CLE extract are primarily responsible for reducing silver ions. The shift of peak from 669 cm −1 to 617 cm −1 in the absorption peaks suggests that the amine components are involved in reducing and stabilizing the nanoparticles. (Fig. 1b,c). Further, the sharp peak at 1645 cm −1 indicated the presence of flavonoids and terpenoids that complied with the reports of the qualitative study of phytochemicals (Table 1). In a similar study, Jayakumar et al. demonstrated the use of FTIR to detect biomolecules for reducing Ag + ions and capping Clerodendrum splendens extract-produced AgNPs 57 . CLE-AgNP was crystalline, mostly spherical, with variable particle size and elemental (Fig. 2a) in nature. The crystallite size of the material from XRD measurements was found to be around 27.67 nm. In addition to the four major XRD peaks in the diffractogram, other unidentified peaks observed might be attributed to the formation of the crystalline biomolecules bound to the surface of the AgNPs. SEM and TEM analysis also confirmed the synthesis of variable size (12-44 nm, mean ≈ 27) and spherical shape AgNPs (Fig. 2b-d). www.nature.com/scientificreports/ Recent studies indicate that smaller nanoparticles are useful for delivering many targets, especially antimicrobial activity, as they have more surface exposure to the bacterial membrane increasing the cell permeability and resulting in cell death 58 . Their small sizes also allow efficient drug accumulation at the target sites with provisions for sustained drug release 59 . The results shown in Fig. 4 demonstrate that CLE-AgNP indeed has anthelmintic efficacy on poultry cestode Raillietina spp. and acts in a dose and time-dependent manner. The individual impact of CLE flower extract at the highest dose (125 µg/ml) was 2.75 ± 0.1 h and 3.21 ± 0.14 h for paralysis and death, respectively, while its anthelmintic activity was enhanced when combined with silver particles (Supplementary  Table S1). Previous studies suggest phytochemicals and their stabilization properties may have a profound colloidal activity causing a synergistic impact on one another when the extract is coated on AgNPs 60 . The most efficacious dose of CLE-AgNP was 125 µg/ml, wherein the initiation of paralysis of parasites occurred after 0.43 h and death after 1.07 h of treatment (Fig. 4). Scanning electron microscopy results have shown discernible topographical mutilation on test worms, while control tapeworm shows smooth and organized rostellum, sucker, and microtriches on the head and strobila portion. As observed in the present investigation, tegumental erosion has altered the host-parasite interface, causing severe nutrient deficiency within the parasite. Apart from the absorption of nutrients, the possible role of spine-like features of the microtriches is holding with its host to maintain its position in the gut. Therefore, disruption of microtriches leads to reduced attachment capacity of the parasites to the host. Similar observations were recorded on incubation of the parasite in Resveratrol, which causes blebbing, swelling of the tegument, loss of spines and distortion of suckers in Raillietina spp. exposed in vitro 61 . Extensive blebbing of Fasciola hepatica surface was seen on treatment with various concentrations of crude ethanolic shoot extract of traditionally used medicinal plants like Alpinia nigra 62 . Other phytocompounds like α-viniferin have been unequivocally responsible for damages in varying degrees on the teguments of cestodes 63 . In a recent study, the tegument and suckers of the tapeworm Raillietina tetragona and the cuticle and lips of the roundworm Ascardia galli both showed rigorous damage on treatment with the roots and rhizomes extract of an ethnomedicinal plant Imperata cylindrica 64 . The tegumental enzymes AcPase and AlkPase are found to be involved in the uptake of certain nutrients, glycogen and lipoprotein in various helminth parasites 65 . Enzymatic alterations in histochemical localization studies also revealed pronounced effects ( Table 2). ATPase is associated with active www.nature.com/scientificreports/ transport, and a significant reduction in its activity in the somatic musculature, and complete absence in the tegument and sub-tegumental layers in 125 µg/ml CLE-AgNP-exposed parasite further indicates the disruption of the energy metabolism pathway in the parasite. In a previous study, following treatment with Genistein, the active component of Flemingia vestita, the tegumental enzymes like AcPase, AlkPase, ATPase and 5'-Nu of Raillietina spp. were found to be decreased many folds 66 . Thus, the observed decrease in the two tegumental phosphatase activities might be related to inhibition or decreased glucose absorption by Raillietina spp., resulting in progressive loss of motor function owing to a deficiency of energy source and, eventually, paralysis and death. In summary, our investigation elucidates a concise, cost-effective, and productive route in the phytosynthesis of AgNPs utilizing the aqueous floral extract of CLE. Further, in vivo, studies are required to understand the mode of action of the drug and its pharmacokinetics. In the future, optimization of the test therapeutic (CLE-AgNP) through pharmaceutical evaluation and application of appropriate feeding strategy by finding out the effects on metabolic precursors and intracellular metabolites can contribute to a seamless transition to financially feasible ethnomedicinal anthelmintic. We believe using green synthesis methods to produce silver nanoparticles from Clerodendrum infortunatum flowers could provide a promising alternative to synthetic anthelmintic drugs for treating helminth infections in domestic fowl.

Materials and methods
Collection of plant material. The fresh and healthy flowers of Clerodendrum infortunatum were collected from the Cooch Behar Panchanan Barma University (CBPBU) campus garden (latitude 26.321796 °N, longitude 89.469329 °E) over a time of 2 months between May to June 2021. Dr. Chaya Deori did the taxonomical evaluation, and the voucher specimen (AC-97296) was kept at the herbarium of the Botanical Survey of India, Eastern Regional Centre, Shillong. We confirm that the collection of plant material complied with relevant institutional, national, and international guidelines and legislation. The appropriate permissions for collecting plant specimens were obtained from the CBPBU administration affiliated with the Government of West Bengal, India.
Preparation of plant extract using flowers of CLE. The flowers were thoroughly washed sequentially with tap and deionized water, air-dried and chopped into small pieces. Chopped flowers (20 g) were added to 100 ml deionized autoclaved water in a beaker and heated at 90 °C in a temperature-controlled water bath for 10 min. The extract was cooled, filtered through Whatman No. 1 filter paper, and kept at 4 °C until used.
Qualitative assessment of phytochemicals. The phytochemical analysis of Clerodendrum infortunatum flower extract was carried out using aqueous extracts using standard procedures outlined in Table 1, with minor modifications to identify the various constituents.

Biosynthesis of CLE-capped AgNP.
For silver nanoparticle synthesis, about 10 mL of CLE flower extract was added to a 90 mL aqueous solution of 1 mM AgNO 3 (Merck Laboratories, India) and kept at room temperature. The color changed from pale yellow to brown, indicating the formation of the AgNPs due to the reaction of flower extracts of CLE with silver metal ions. Control was maintained without adding flower extract in the silver nitrate solution that showed no color changes. The purified AgNPs were obtained by removing the extract by centrifuging the suspension thrice at 15,000 rpm for 20 min, followed by washing it twice with double sterilized water.
Characterization studies of AgNP. Visible colour changes preliminarily determined the bio-reduction process of silver ions in solution and later monitored using UV-visible absorption spectroscopy (Hitachi U-2900). The synthesized AgNPs were freeze-dried, powdered, and used for XRD analysis. The dried powder was mixed with potassium bromide at the ratio of 1:100, and the results were recorded using FTIR (Jasco FR/ IR-6300). The CLE-AgNPs were critical point dried, placed over a carbon tape onto an SEM stub, platinum coated, and viewed under a scanning electron microscope (Zeiss EVO-18) at a voltage of 15 kV to assess the surface morphology of the biogenic AgNPs. Transmission electron microscopy (JEOL JEM-2100 HR) was used to understand the size and morphology of AgNPs. Cestoda; Order: Cyclophyllidea; Family: Davaineidae) were collected from the intestine of freshly sacrificed domestic fowl (Gallus gallus domesticus L.) from local abattoirs in Cooch Behar and maintained in 0.9% PBS at 37 ± 1 • C in an incubator. Control parasites were maintained in 0.9% PBS (pH 7.2 ) at 37 ± 1 • C , whereas for treatment, live worms were directly incubated in different concentrations of test treatment ( 25,50,75, 100, 125 µg/ml) in separate Petri dishes, both for the CLE flower aqueous extract and CLE-AgNPs. Similarly, treatment was also performed with Genistein at a dose of 125 µg/ml of PBS as a broad-spectrum reference drug. Six replicates for each set of incubation mediums were prepared, and the time taken to attain the paralytic state and death was recorded. Mortality of parasites was confirmed by removing treated parasites from the test medium and dipping them in slightly lukewarm water, which was indicated by the cessation of all signs of movement. The control and CLE-AgNP treated parasites were retrieved from the respective incubation mediums and processed for histochemical and electron microscopic studies.

Collection of parasites and in vitro
Scanning electron microscopy of the parasite. The parasites were fixed in 10% neutral buffered formalin for 24 h immediately after paralysis. After fixation, the sample was rinsed in PBS and dehydrated with acetone grades ranging from pure dry acetone to escalating degrees of acetone. The specimens were then critical- www.nature.com/scientificreports/ point dried using liquid carbon dioxide as the transitional fluid, which was put on a metal stub and coated with platinum in a fine-coat ion sputter, JFC-1100 (JEOL). The specimens were then viewed using a Zeiss EVO-18 (Special edition) scanning electron microscope at an accelerating voltage of 10-15 kV.
Histochemical studies. The tegumental enzymes were investigated histochemically using duly processed frozen sections cut at a thickness of 10-12 µm in a Leica CM 3050S cryostat. AcPase activity was detected in cold formol calcium fixed specimens following the modified lead nitrate method 67 , using sodium β-phosphoglycerate as the substrate, where a brownish precipitate on tegumental sections indicates the sites of AcPase activity. The modified coupling azo-dye method assessed AlkPase activity at room temperature (17-20 °C). The calcium method of Pearse was implemented to detect the localization of ATPase activity, where adenosine triphosphate was used as the substrate, and the enzyme activity was determined by observing blackish-brown deposits 67 . To observe 5'-Nu, the lead method of Wachstein and Meisel was employed using adenosine monophosphate as a substrate 68 . The histological sections were viewed using Carl Zeiss Axiolab 5 Phase Contrast Microscope.
Ethics approval. We confirm that any aspect of the work covered in this manuscript does not involve ethical approval.

Data availability
The data used to substantiate the findings of this study are included in the article; however, the raw data is also available from the corresponding author upon request.